An SCN1A gene missense variant in a Chinese Tujia ethnic family with genetic epilepsy with febrile seizures plus

Genetic epilepsy with febrile seizures plus (GEFSP) is a familial epileptic syndrome that is genetically heterogeneous and inherited in an autosomal dominant form in most cases. To date, at least seven genes have been reported to associate with GEFSP. This study aimed to identify the disease-causing variant in a Chinese Tujia ethnic family with GEFSP by using whole exome sequencing, Sanger sequencing, and in silico prediction. A heterozygous missense variant c.5725A>G (p.T1909A) was identified in the sodium voltage-gated channel alpha subunit 1 gene (SCN1A) coding region. The variant co-segregated with the GEFSP phenotype in this family, and it was predicted as disease-causing by multiple in silico programs, which was proposed as the genetic cause of GEFSP, further genetically diagnosed as GEFSP2. These findings expand the genetic and phenotypic spectrum of GEFSP and should contribute to genetic diagnoses, personalized therapies, and prognoses.


. Introduction
Genetic epilepsy with febrile seizures plus (GEFSP, also called GEFS+), previously known as generalized epilepsy with febrile seizures plus, was first defined by Scheffer and Berkovic as an inherited epileptic syndrome with genetic and phenotypic heterogeneity (1,2). It is a familial epilepsy syndrome clinically characterized by various types of seizures, including fever-associated and afebrile seizures (3,4). GEFSP includes a wide range of subtypes with varying prevalence, of which the estimated prevalence of febrile seizures (FS) is 3-4%, and the overall prevalence is unclear (5,6). The most common phenotype in the GEFSP pedigrees is FS, which occurs between the age of 3 months and 6 years, followed by febrile seizures plus (FS+), in which episodes with fever persist beyond 6 years or afebrile seizures occur (7). The diagnosis is based on the occurrence of two or more family members that show phenotypes in the spectrum, including FS, FS+, FS/FS+ with various seizures (such as absence, atonic, myoclonic, and partial seizures), and myoclonicatonic epilepsy, along with Dravet syndrome (8)(9)(10). The inheritance patterns include autosomal dominant, autosomal recessive, and complex inheritance in which several genes are involved, accompanied by possibly environmental contributions. In pedigrees with monogenic variants, most of them follow autosomal dominant patterns, and the minority .
/fneur. . abides by autosomal recessive traits. In some small families, the most common pattern may be complex inheritance (11,12). To date, at least 11 genetic loci are recorded for GEFSP in the Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/). Variants in the critical regions of at least seven genes, including the sodium voltage-gated channel beta subunit 1 gene (SCN1B), the sodium voltage-gated channel alpha subunit 1 gene (SCN1A), the gamma-aminobutyric acid type A receptor subunit gamma2 gene (GABRG2), the gamma-aminobutyric acid type A receptor subunit delta gene (GABRD), the syntaxin 1B gene (STX1B), the hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 1 gene (HCN1), and the hyperpolarizationactivated cyclic nucleotide-gated potassium and sodium channel 2 gene (HCN2), were reported to be responsible for GEFSP (13)(14)(15)(16)(17)(18)(19). Of these, the SCN1A gene is the most clinically relevant and most frequently reported disease-causing gene for the GEFSP spectrum, and ∼11% of reported pedigrees were caused by its variants (20).
In this study, we identified a heterozygous missense variant c.5725A>G (p.T1909A) in the SCN1A gene as the causative variant in a Chinese Tujia ethnic family with GEFSP.
. Materials and methods

. . Participators and clinical evaluations
A non-consanguineous Chinese Tujia ethnic family located in the west of Hunan province was recruited from the Third Xiangya Hospital, Central South University (Changsha, China). The GEFSP diagnosis was based on clinical features, family history, electroencephalography (EEG), and genetic testing. The medical history of the patients was collected. Routine physical examinations and EEG were performed on the proband. All individuals had signed the written informed consent before the peripheral venous blood samples were acquired. The approval of this study was received from the Institutional Review Board of the Third Xiangya Hospital, Central South University, Changsha, China.

. . DNA extraction and exome capture
The genomic DNA (gDNA) was extracted from peripheral venous blood samples by using the standard phenol-chloroform extraction method as previously described (21). The gDNA samples of II:1 and III:1 ( Figure 1A) were randomly fragmented to 150 bp-250 bp using Covaris technology and prepared for whole exome sequencing (WES). The ends of DNA fragments were repaired, and the "A" base was ligated at the 3 ′ -end of each strand. Adapters were added to both ends of the end-repaired DNA for PCR amplification and further sequencing. The products were then purified and hybridized to the exome array. Hybridized fragments were used for circularization, and non-specific fragments were cleaned out (22). DNA nanoballs were produced by rolling circle amplification. The qualified captured library was loaded on the BGISEQ-500 sequencing platform, and sequencing-derived raw image files were analyzed by BGISEQ-500 base-calling software, performed by the BGI-Shenzhen (Shenzhen, China).

. . Clinical findings
The proband (III:1) was a 22-year-old female with unremarkable spontaneous vaginal delivery and development. Her first seizure occurred at the age of 2 years when she suffered from an upper respiratory tract infection and had a fever of ∼38 • C. After that, she had recurrent episodes when she caught a fever or felt nervous. She has suffered from attacks of afebrile seizures (AFS) since the age of 14 years. The episodes can manifest as moderate generalized tonic-clonic seizures, with a mean duration of 2-7 min, and focal impaired awareness .
/fneur. . seizures, with a mean duration of 1-1.5 min. The ambulatory EEG showed interictal epileptiform discharges: bilateral slow waves, sharp waves, and sharp-and-slow wave complexes (41). Irregularly antiepileptic drug lamotrigine treatment not adhering to medical advice was applied, and the efficacy was not good. The self-reported medical history of her father (II:1) revealed fever-associated seizures at 7 months and spontaneous remission before the age of 14 years. Focal impaired awareness seizures began at the age of 7 years, and no AFS were claimed.
Detailed features of patients in this family are presented in Table 1.

. . Genetic analysis
Overall, the WES of the two patients generated an average of 203.59 million effective reads. Of these, ∼99.95% were mapped to .
/fneur. .  by bioinformatics (Table 2). Sanger sequencing corroborated the variant in the patients (II:1 and III:1, Figure 1B) and revealed the absence of the variant in the proband's unaffected mother (II:2, Figure 1C). Conservation analysis showed the high conservation of the mutated p.T1909 residue among 10 species (Figure 1D), and the conformation change caused by the variant was shown in Figure 2. Based on the above evidence, the variant c.5725A>G (p.T1909A) in the SCN1A gene appears to be accountable for the GEFSP in this family. According to ACMG guidelines, the SCN1A gene variant c.5725A>G (p.T1909A) was classified as a "likely pathogenic" (PS1 .

Discussion
GEFSP is a familial epileptic syndrome with remarkable genetic and phenotypic heterogeneity (42). It was first described in a large Australian family having an unusual concentration of generalized epilepsy and FS (43). Since the SCN1B gene was reported as the first causative gene of GEFSP in 1998, a large number of GEFSP-related .
/fneur. . variants have been identified in seven pathogenic genes, in which the SCN1A gene is the most frequently reported and most clinically relevant one (44).
In this study, we identified the SCN1A missense variant c.5725A>G (p.T1909A) in two members of the Chinese Tujia ethnic family via WES and Sanger sequencing, which was found to co-segregate with the phenotype in the family. The proband had AFS and uncontrolled seizures in adulthood, and her father had self-limited seizures, in favor of the phenotypic heterogeneity of GEFSP, genetically diagnosed as GEFSP2.
The SCN1A gene, located on chromosome 2q24.3, includes 27 exons, encoding the protein sodium voltage-gated channel alpha subunit 1 (Na v 1.1) with 2,009 amino acids (∼229 kDa) (45). The Na v 1.1 includes four homologous domains (I-IV), each containing six alpha-helical transmembrane segments (S1-S6), in which the S4 is a voltage sensor, and the S5 and S6 form the ion-selective pores ( Figure 1E) (46). It is highly expressed in the brain, especially the prefrontal cortex, and forms the voltage-dependent sodium channel with beta subunits as one of the primary sodium channels in the central nervous system (http://biogps.org/), which plays a crucial role in the initiation and propagation of action potentials in neurons (47,48).
To date, more than 2,584 variants in the SCN1A gene have been recorded, and most are deemed responsible for GEFSP phenotypes (HGMD, http://www.hgmd.cf.ac.uk/ac/index. php). The c.5725A>G variant was located at the exon 27 of the SCN1A gene. It was absent in public databases including dbSNP, the 1000 Genomes Project, NHLBI ESP6500, ExAC, gnomAD, ChinaMAP, ClinVar, and the in-house BGI exome database. The variant was predicted as deleterious or probably damaging by multiple in silico programs including MutationTaster, PROVEAN, SIFT, PolyPhen-2, and MutationAssessor. The threonine at position 1,909 was phylogenetically conserved among varied species from fruit flies to human beings. The polar hydrophilic to non-polar hydrophobic residue change may affect the tertiary structure and impact normal function (49). The p.T1909A variant is located in the cytoplasmic region of the Na v 1.1 C-terminal domain, which contains binding sites of interaction proteins and plays an important role in channel inactivation (50).
Functional studies indicated that different SCN1A variants alter sodium channel properties and functions in different ways and cause distinctive effects on the sodium channel activity, thus affecting the selection of antiepileptic drugs (52). Both gain-of-function (GOF) and loss-of-function (LOF) effects could be responsible for SCN1A-associated epilepsies. In human tsA201 cells with p.R1648C or p.F1661S variant, a small non-inactivating persistent inward current during depolarization was reported to lead to neuron hyperexcitability, which exhibited a GOF effect in the sodium channels (53). GOF-related epilepsy can usually be relieved by commonly prescribed antiepileptic drugs that inhibit sodium channels, such as carbamazepine and phenytoin (54). SCN1A LOF variants mainly impair bipolar GABAergic inhibitory interneurons and lead to diminished inhibition (55). The application of GABA transaminase inhibitors such as valproic acid or GABA receptor-positive allosteric modulators, such as pentobarbital, appears to be effective, while the sodium channel blocker may provoke seizure aggravation (56). In human tsA201 cells, the SMEI-associated p.T1909I variant exhibited a mixture mechanism of GOF and LOF, with increased persistent current and reduction of current density (57). In this study, disease symptom remission and seizure frequency reduction after lamotrigine application in the proband were unsatisfactory. Due to the same biophysical property change (polar hydrophilic to non-polar hydrophobic) of p.T1909I and p.T1909A variants, the same mechanism may be shared. Limited response to medication may be due to the use of a single sodium channel blocking antiepileptic drug. The prescription of GABA transaminase inhibitors or GABA receptor-positive allosteric modulators might be beneficial for the sufferers.
Genetic deficient animal models verified the important role of the SCN1A gene in epilepsy development. The drosophila SCN1A p.K1270T variant knock-in model showed that when the temperature rose, the deactivation threshold for persistent sodium currents reversibly shifted to a more negative voltage, causing sustained depolarizations in GABAergic inhibitory interneurons and leading to reduced inhibitory activity in the brain (58). Heterozygous (Scn1a +/− ) mice had spontaneous seizures and occasional deaths beginning on postnatal day 21, attributed to haploinsufficiency of Na v 1.1 channels, and homozygous Scn1a knockout (Scn1a −/− ) mice developed severe ataxia and seizures and died on postnatal day 15, corresponding to the LOF effect (59). In a bacterial artificial chromosome transgenic SCN1A p.R1648H variant mouse model, experiments showed a delayed recovery of channel inactivation only in inhibitory neurons, suggesting the cell type-dependence of SCN1A mutation and the p.R1648H variant leading to a reduction in inhibitory neurons excitability (60).

. Conclusion
In summary, a missense variant c.5725A>G (p.T1909A) in the SCN1A gene was identified in a Chinese Tujia ethnic family with GEFSP, further classified as GEFSP2. The different phenotypes of the same variant in the family show the heterogeneity of GEFSP. These findings confirmed the SCN1A-associated GEFSP, which may expand the genetic and phenotypic spectrum of GEFSP and improve genetic diagnoses, personalized therapies, and prognoses. The combination of WES and Sanger sequencing efficiently provided a timely diagnosis and indicated management for those with clinically and/or genetically suspected GEFSP. Additional in vitro studies and the establishment of genetically deficient animal models to explore the functional effect of human SCN1A variants may help to further illuminate potential pathogenic mechanisms.

Data availability statement
Data of this project can be accessed after an approval application to the China National Genebank (CNGB, https://db. cngb.org/cnsa/). Please refer to https://db.cngb.org/, or email: CNGBdb@cngb.org for detailed application guidance. The accession code CNP0004397 should be included in the application.

Ethics statement
The studies involving human participants were reviewed and approved by the Institutional Review Board of the Third Xiangya Hospital, Central South University, Changsha, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.